This invention relates to the method of separating the three major populations of leukocytes, namely, lymphocytes (lymph), monocytes (mono), and granulocytes (gran) populations of white blood cells from a white blood cell histogram which has been derived from automated cell counters which electronically count and size cells employing the well known impedance principles.
To differentiate and identify white cell types, the white blood cells are differentially lysed into separate size classes. The size of each cell is then plotted against a frequency of occurrence into a composite histogram. This histogram contains all the types of white blood cells as is illustrated, for example, in U.S. Pat. No. 4,485,175. The histogram is then analyzed to separate out the three major types of white blood cells, namely, lymph, mono and gran populations of the white blood cells.
A number of difficulties have been involved in separating the three populations from the composite histogram which include the fact that the middle population can be extremely close to the left or lymph population of the histogram and sometimes a minimum point between these two populations cannot be found. The middle population of the histogram is also relatively small compared to the two surrounding populations constituting from 5 to 10% of the total histogram. The right gran population is extremely broad causing it to interfere with the mid population and the shape of the histogram, between the middle population and the gran population, is flat making it difficult to establish a stable minimum point therebetween. The gran population can also skew both left or right which makes the use of a log-normal curve fit very difficult. In addition, each population can vary in area from zero and normal curve fits tend to blow up for very small areas.
In the past, fixed discriminators have been provided setting thresholds levels between the three major populations. The counts are made using a large number of count channels which are divided by the discriminators having fixed threshold positions which do not change once they are set. Once the threshold positions are set an entire series of histograms is fed thereto. The nature of the middle population simply does not lend itself to the use of fixed discriminators which tend to miss the middle populations in any type of controls applied thereto. In addition, changes in temperatures or lyse concentrations affect the size distribution and by varying these parameters the location or width of each population will change. Therefore, in evaluating a histogram with discriminators having fixed thresholds, much thresholds will not factor in the varying parameters and will provide inaccurate counts.
The present invention is directed to overcome the aforesaid difficulties.